Elucidating Pattern Formation in Anabaena: A Genetic Approach

丝状蓝藻中的水芽藻，对氮的缺乏作出反应，沿着丝状半规则间隔的细胞（异囊）发育，专门用于固定氮。异囊的模式是由发育和成熟的异囊产生的抑制场发起和维持的。沃尔克博士已经分离出了在发育早期就有缺陷的突变体并发现了能补充这些突变体的微粒。为了研究灯丝发育过程中基因表达的空间方面，他开发了一种促进荧光素酶（lux）转录融合及其维持的载体。他建议：1。进一步发展和表征lux用于监测单个细胞的基因表达。2. 确定在发育早期表达的基因，特别是在注定成为异囊的细胞中。候选启动子将被熔合成流光和沿着灯丝的发光，并在整个开发过程中进行监测。3. 将携带成功启动子/lux融合的水藻进行转座子诱变和反义RNA表型诱变，寻找调控启动子表达的基因。这样的基因将通过改变的光发射来识别。Wolk博士将专注于通过在所有细胞中诱导发光来对N-递减作出反应的突变体，而不仅仅是在发育中的异囊中。4. 克隆影响启动子表达的基因，并通过将它们融合到lux中，使它们在时间和空间上的表达本地化。高等真核生物从单个细胞发展到多种类型和功能的细胞集合，但原核生物的不均匀分化是罕见的。Wolk博士建议利用丝状蓝藻（Anabaena）的分化，在一个简单的原核生物中研究这些过程，在那里可以利用强大的遗传技术。

Anabaena in a filamentous cyanobacterium that responds to nitrogen deprivation by elaborating along the filament semi-regularly spaced cells (heterocysts) specialized for N2-fixation. The pattern of heterocysts is initiated amd maintained by an inhibitory field emanating from developing and mature heterocysts. Dr. Wolk has isolated mutants that are defective early in development and have found cosmids that complement certain of these mutants. To study spatial aspects of gene expression in the developing filament, he has developed a vector that facilitates transcriptional fusions to luciferase (lux) and their maintenance in Anabaena. He proposes to: 1. Further develop and characterize the use of lux to monitor gene expression from individual cells. 2. Identify a gene that is expressed early in development, specifically in cells fated to become heterocysts. Candidate promoters will be fused to lux and light emission along the filament monitored through the course of development. 3. Subject Anabaena carrying the successful promoter/lux fusions to transposon mutagenesis and antisense RNA phenotypic mutagenesis to search for genes that regulate the expression of the promoter. Such genes will be identified by altered light emission. Dr. Wolk will focus on mutants that respond to N- stepdown by inducing light emission in all cells rather than only in developing heterocysts. 4. Clone genes that affect the expression of the promoter and, by fusing them to lux, localize their expression in time and space. Higher eukaryotes develop from single cells to collections of cells of varied types and functions, but inhomogeneous differentiation in prokaryotes is rare. Dr. Wolk proposes to exploit the differentiation of the filamentous cyanobacterium, Anabaena, to study these processes in a simple prokaryote where powerful genetic techniques can be utilized.